9. Cell-matrix adhesion structures were studied in the experiment described belo
ID: 255290 • Letter: 9
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9. Cell-matrix adhesion structures were studied in the experiment described below. Fibroblasts were cultured on glass coverslips. The cells were fixed and immunostained with fuorescently labeled phalloidin (green) and an anti-vinculin antibody (red). The samples were visualized on a fluorescence microscope (image shown below-be sure to view on computer screen in color). What do the green-stained and red-stained structures each correspond to in the cell? Describe this at the level of detail that was covered in Chapter 15. (5 pts)Explanation / Answer
cell matric adhersion structures were stained and explained as,
Fibroblasts were stained using antibodies anti- vinculin with phalloidin. Stained cells were examined by fluorescence microscopy. The appearance of the cells is similar to the appearance of the cell on the uncoated slide shown in fig. Note the focal staining for vinculin at the cell periphery.
Fibroblast on partially degraded collagen stained for vinculin. Vinculin is distributed diffusely over the cell surface. Actin filaments are not evident, although phalloidin reactivity can be seen at the cell periphery where the cell appears to be in contact with the underlying substratum
When cells on uncoated slides were examined by confocal fluorescence microscopy adhesion sites were defined in the well spread cells by intense, focal staining for vinculin. Specifically, there was localized vinculin staining around the cell periphery at sites where the cell appeared to be in contact with the substratum.
Thus this fig confirms that actin filaments were associated with vinculin. The staining patterns for both vinculin were much different in the cells maintained on partially degraded collagen. On the partially degraded collagen substrate, vinculin was diffusely present over the whole cell surface. No actin filaments were visualized in these cells.
View showing vinculin staining at the cell periphery and actin filaments terminating in association with vinculin. Fibroblast on partially degraded collagen stained for vinculin. Vinculin is distributed diffusely over the cell surface.
Actin filaments are not evident, although phalloidin reactivity can be seen at the cell periphery where the cell appears to be in contact with the underlying substratum.
1472 VARANI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY part of the molecular complex that directly interacts with
extracellular collagen. Results virtually identical to those obtained with vinculin were obtained.
In a final set of experiments, an antibody to smooth muscle a-actin was used in place of phalloidin to confirm that phalloidin staining of the visible stress fibers was actually due to the presence of the actin filaments.
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